A plasma coagulation assay for an activated protein C-independent anticoagulant activity of protein S.

To study the physiological importance of the activated protein C (APC)-independent anticoagulant activity of protein S, we developed an assay specific for this activity. The ability of protein S to prolong the clotting time in an APC-independent way was expressed as the ratio of the clotting time in a plasma sample divided by the clotting time in the presence of a polyclonal antibody against human protein S (both after 1:1 dilution in protein S-C4BP deficient plasma). The mean protein S-dependent anticoagulant ratio (PSdAR) was 1.53+/-0.18 in 34 healthy controls, and was significantly lower in 16 heterozygous protein S deficient patients (PSdAR=1.15+/-0.09). In plasmas from patients under oral anticoagulant therapy the mean PSdAR was within the range of the control population (1.50+/-0.18). The mean total protein S antigen level in these plasmas was 58%, suggesting a higher specific APC-independent anticoagulant activity of protein S in these patients than in normals. This functional protein S assay can be used for the laboratory diagnosis of protein S deficiency, and to study the mechanism of the APC-independent anticoagulant activity in plasma.

Characterization of mini-protein S, a recombinant variant of protein S that lacks the sex hormone binding globulin-like domain.

Protein S is a vitamin K-dependent glycoprotein involved in the regulation of the anticoagulant activity of activated protein C (APC). Also, an anticoagulant role for protein S, independent of APC, has been described. Protein S has a unique C-terminal sex hormone binding globulin (SHBG)-like domain that represents about half of the molecule. To define the role of this domain in APC cofactor activity and in binding to C4b-binding protein (C4BP), we have constructed a recombinant protein S molecule of N-terminal residues 1-242 that lacks the SHBG domain (mini-protein S). A panel of monoclonal antibodies directed against the N-terminal region of protein S recognized plasma-derived protein S, wild-type recombinant protein S and mini-protein S with similar affinities, whereas a monoclonal antibody that recognizes an epitope in the SHBG domain did not detect mini-protein S. Mini-protein S did not bind to C4BP in a solid-phase binding assay, and the cofactor activity of mini-protein S was not inhibited by preincubation with C4BP. In a plasma coagulation assay, the cofactor activity of mini-protein S was lower than wild-type or plasma-derived preparations. In contrast, no difference in APC cofactor activities was observed when the preparations were tested in purified systems that monitor the APC-mediated degradation of factors Va or VIIIa. In conclusion, we constructed a protein S molecule that fails to bind C4BP and still displays cofactor activity for APC. This confirms the role of the C-terminal SHBG region in C4BP binding and demonstrates that N-terminal residues 1-242 are sufficient for the expression of APC cofactor activity in a system using purified components. In plasma, however, the C-terminal SHBG region plays a role in the expression of optimal APC cofactor activity.

The interaction of protein S with the phospholipid surface is essential for the activated protein C-independent activity of protein S.

Protein S is a vitamin-K dependent glycoprotein involved in the regulation of the anticoagulant activity of activated protein C (APC). Recent data showed a direct anticoagulant role of protein S independent of APC, as demonstrated by the inhibition of prothrombinase and tenase activity both in plasma and in purified systems. This anticoagulant effect of protein S can be explained either by a direct interaction of protein S with one of the components of the complexes and/or by the interference with the binding of these components to phospholipid surfaces. During our investigation we noted that protein S preparations purified in different ways and derived from different sources, expressed discrepant APC cofactor and direct anticoagulant activity. In order to elucidate these differences and to study the mechanism of the APC-independent activity of protein S, we compared the protein S preparations in phospholipid-binding properties and anticoagulant activity. The dissociation constant for the binding of protein S to immobilized phospholipids ranged from 7 to 74 nM for the different protein S preparations. APC-independent inhibition of both prothrombinase and tenase activity performed on phospholipid vesicles and in plasma showed a strong correlation with the affinity for phospholipids. The APC-independent activity could be abolished by monoclonal antibodies that were either calcium-dependent and/or directed against epitopes in the Gla-region of protein S, suggesting that the protein S-phospholipid interaction is crucial for the APC-independent anticoagulant function of protein S. Protein S preparations with a low APC-independent activity expressed a high APC-cofactor activity suggesting that the affinity of protein S for phospholipids is of less importance in the expression of APC-cofactor activity of protein S. We conclude that high affinity interactions of protein S with the membrane surface are essential for the direct anticoagulant activity of protein S and we suggest that inhibition of the prothrombinase and the tenase complex by protein S is a consequence of the occupation of the phospholipid surface by protein S molecules.

von Willebrand factor as a regulator of intrinsic factor X activation.

Factor VIII is an important cofactor in the intrinsic activation of factor X. To function effectively as a cofactor, factor VIII must be activated. In plasma, factor VIII circulates in a complex with von Willebrand factor, and although thrombin can activate complexed factor VIII, the activation by activated factor X is inhibited by von Willebrand factor. In this study, the effect of von Willebrand factor on the generation of factor Xa by the factor IXa-VIII complex was investigated. Purified human factors VIII, IXa, and X were incubated on human umbilical vein endothelial cells or phospholipid vesicles in the presence of calcium ions, and the generation of factor Xa was followed. In the presence of von Willebrand factor, a prolonged lag-phase and a dose-dependent inhibition of factor X activation was observed. These effects were not observed when von Willebrand factor was preincubated with a monoclonal antibody directed against von Willebrand factor that blocks factor VIII binding. When factor VIII was activated with thrombin before the incubation, neither the monoclonal antibody nor von Willebrand factor had an effect on the rate of factor X activation. Preincubation of endothelial cells with the monoclonal antibody resulted in a somewhat higher rate of factor X activation. When endothelial cells from a patient with von Willebrand's disease type I were used, preincubation of the monoclonal antibody had no effect on the rate of factor X activation. We conclude that von Willebrand factor on the surface of endothelial cells can modulate the intrinsic factor X activation. This effect is greatly enhanced, however, by the addition of exogenous von Willebrand factor.(ABSTRACT TRUNCATED AT 250 WORDS)